Insulin-like growth factors (IGFs) are mitogenic peptide hormones that play important roles in the growth and differentiation of vertebrates. IGFs are translated as a prepropeptide, which can be divided into an N-terminal prepeptide (which is also called a signal or leader peptide), a B to D domain polypeptide (which contains peptides from B, C, A and D domains), and a C-terminal E-domain peptide (which is also called a trailer peptide). The IGF signal and E peptides are proteolytically removed from the B to D polypeptide during protein maturation (Shamblott and Chen (1992), Proc. Natl. Acad. Sci. USA, 89:8913-8917). For this reason, the B to D domain polypeptide is also called "the IGF mature peptide".
So far, the functions of the IGF signal and E peptides are unknown. However, it is well known in the art that a signal peptide may facilitate the transport of protein out of the cell membrane. As for the E peptide, since the E peptide is a part of the IGF preproprotein and may actually be secreted as a part of an intact prohormone, it is possible that the E peptide may affect the processing, transport, or secretion of the mature peptide. It is also possible that the E peptide may indirectly or directly modulate the degradation, receptor interaction, or binding protein interactions of the mature peptide.
There are two kinds of IGFs, namely, IGF-I and IGF-II. IGF-I and IGF-II share high homology of protein folding structure and similar growth promotion effects, even though they are mediated by different IGF receptors (i.e., IGF-I is mediated by a tyrosine kinase receptor, whereas IGF-II is mediated by a mannose-6-phosphate receptor). IGF-I is a 70 amino acid polypeptide which mediates the growth-promoting actions of growth hormone as well as having important local paracrine and autocrine roles in multiple organs (Kavsan et al. (1993), DNA and Cell Biology, 12:729-737).
In mammals, the mature form of IGF-II is a neutral protein consisting of 67 amino acid residues with three disulfide bonds. IGF-II is a single-chain polypeptide that contains a NH.sub.2 -B-C-A-D-COOH domain. The signal peptide and E domain are removed in mature peptide. The cDNA sequences of IGF-II have been reported highly conserved in different mammals, including chickens, sheep, mice, humans, and rats. The mammalian IGF-II is primarily produced in liver during prenatal development under the control of placental lactogen (Gray et al. (1987), DNA, 6:283-295). Because of its role as a key component regulating fetal growth, IGF-II is also called a "fetal growth factor".
IGF-II has a complex gene structure. In humans, IGF-II gene consists of 10 exons and spans about 30 kb of DNA. The DNA sequence encoding the mature IGF-II polypeptides in humans is contained within exons 8, 9, and 10. In rat and mouse, IGF-II genes consist of 6 exons and span about 12 kb of DNA and the DNA sequence encoding the prepropeptide of rat or mouse IGF-II is contained within exons 4, 5, and 6.
Up until now, the gene structure and protein function of IGF-II in fish have remained unidentified. IGF-II like peptides have been reported present in the insulin cells of the elasmobranchian endocrine pancreas of fish (Reinneke et al. (1994), Histochemistry, 102:365-371). However, whether IGF-II is functioned as autocrine or paracrine is so far unclear. In addition, although so far two IGF-II cDNAs have been discovered in the liver of two fish species: Sparus aurata (Duguay et al. (1996), J. Mol. Endocrinol., 16:123-132) and rainbow trout (Shamblott and Chen (1993), supra), there has been no report or study relating to the expression of fish IGF-II in cells as well as the physiological activity of fish IGF-II in vivo. Furthermore, there has been no report with regard to the findings of IGF-II cDNA in tilapia (Oreochromis mossambicus), and the production of a biologically active IGF-II polypeptide from a tilapia cDNA.
The invention to be presented below describes the cloning and sequencing of a fish IGF-II gene and the production of the biologically active fish IGF-II recombinant polypeptides. The establishment of a gene expression system is important because it provides a multifaceted channel for studies of the functional activity of the expressed proteins. In this case, the successful development of a gene expression system for fish IGF-II is especially important because it not only allows for the production of high quality fish IGF-II protein which facilitates the antibody production for immuno-histochemical study (e.g., RIA, ELISA, etc.), but also provides large quantities of fish IGF-II protein for studies of the physiological functions of IGF-II in fish, particularly in determining the stimulatory effects on growth of juvenile fish.